LA419, a Novel Nitric Oxide Donor, Prevents Pathological Cardiac Remodeling in Pressure-Overloaded Rats Via Endothelial Nitric Oxide Synthase Pathway Regulation
Reduced endogenous NO production has been described in cardiovascular disorders as cardiac hypertrophy and heart failure. The therapy with conventional nitrates is limited by their adverse hemodynamic effects and drug tolerance. The novel NO donor LA419 has demonstrated important antithrombotic and anti-ischemic properties without those adverse effects. The aim of this study was to evaluate the effect of LA419 chronic treatment on cardiac hypertrophy development in a progressive model of left ventricular hypertrophy. Rats were randomly divided into 6 groups: sham and clip (euthanized 7 weeks after aortic stenosis), sham+vehicle, sham+LA419, clip+vehicle, and clip+LA419 (euthanized 14 weeks after the surgery and treated with vehicle or 30 mg/kg of LA419 once left ventricular hypertrophy was established). LA419 treatment for 7 weeks induced a marked reduction in the heart:body weight ratio (4.10±0.28 and 3.38±0.06 mg/g in clip+vehicle versus clip+LA419; P<0.001) and left ventricular diameter (11.96±0.25 and 9.90±0.20 mm in clip+vehicle versus clip+LA419; P<0.001) without modifying the high blood pressure observed in stenosed rats. Histological analysis revealed that LA419 attenuated myocardial and perivascular fibrosis observed in rats with pressure overload for 14 weeks. In addition, LA419 treatment restored endothelial NO synthase and caveolin-3 expression levels, enhanced the interaction between endothelial NO synthase and its positive regulator the heat shock protein 90, and re-established the normal cardiac content of cGMP in stenosed rats. Thus, LA419 prevented the progression to maladaptative cardiac hypertrophy in response to prolonged pressure overload through a mechanism that involved the re-establishment of the endothelial NO synthase signaling pathway.
Left ventricular (LV) hypertrophy (LVH) is a strong independent predictor of cardiovascular events. Patients with LVH have an increased risk of stroke, coronary heart disease, congestive heart failure, and sudden cardiac death.1,2 LVH is usually considered to be initially adaptive by normalizing wall stress. However, chronic pressure overload leads eventually to contractile depression, ventricular dilatation, interstitial cardiac fibrosis, and the development of heart failure.3,4 The mechanisms involved in LVH development and its transition to heart failure are undoubtedly multifactorial and are the subject of intense investigation.
A reduced endogenous NO production has been described in many cardiovascular disorders, including cardiac hypertrophy and heart failure.5 Moreover, it has been demonstrated that both exogenous NO administration and endogenous NO production are able to prevent cardiac hypertrophy development.6,7 In addition, patients with severe pressure-overload hypertrophy showed marked improvement in diastolic function after acute administration of classical NO donors,8 and an increase in LV function has been demonstrated recently using low doses of the NO donor S-nitrosoglutatione.9 Unfortunately, the use of conventional nitrates is limited by their adverse hemodynamic effects (hypotension) and drug tolerance development. Therefore, there is a considerable interest in the development of new NO donors that could offer a prolonged half-life to use in chronic treatment without those negative effects. LA419 is a new neutral sugar organic nitrate with a protected thiol group in its molecular structure that, at therapeutic doses, has no effect on systemic blood pressure and has important antithrombotic10,11 and anti-ischemic properties, exceeding those observed with the standard NO donor isosorbide-5-mononitrate.12
Based on this evidence, the present study was designed to evaluate the effect of chronic treatment with the novel NO donor LA419 on cardiac remodeling associated with cardiac hypertrophy development in rats subjected to aortic stenosis and to investigate its effect on the endogenous NO pathways with special attention to the constitutive NO synthases, endothelial NO synthase (eNOS) and neuronal NO synthase (nNOS) isoforms, and the allosteric regulators, the chaperone heat shock protein 90 (Hsp90) and caveolin-3.
An expanded Methods section can be found in the online supplemental data available at http://hyper.ahajournal.org.
All of the procedures were performed in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Bioethical Committee of Consejo Superior de Investigaciones Científicas.
LVH Model and Experimental Design
LVH was induced by the aortic stenosis technique using a silver clip (0.3-mm aperture).13 Established LVH was obtained 7 weeks after surgery. Each operated rat was paired with a sham-operated rat without any clip implanted. The mean arterial pressure was determined via the catheterized left carotid artery.
The study was performed on 126 adult male Sprague-Dawley rats. A scheme of the experimental design is shown in Figure 1. Animals were randomly distributed in 6 experimental groups: (1) sham (n=14); (2) clip (n=12; both of these groups were euthanized 7 weeks after the surgery); (3) sham+vehicle (n=28); (4) sham+LA419 (n=16); (5) clip+vehicle (n=36); and (6) clip+LA419 (n=20; the latter 4 groups were euthanized 14 weeks after the surgery). LA419 (30 mg/kg) was administered in the drinking water14 7 weeks after the surgery.
Histological examination was performed using 3 to 13 animals in each experimental group. Hematoxylin/eosin stain was used for morphological analysis to assess morphological changes, and Masson’s trichrome stain was used for detection of interstitial and perivascular collagen. Using a light microscope (Olympus Bx40), examinations of the slides were performed in a blind fashion without knowledge of the treatments given. To estimate stereological parameters, a square lattice test system was inserted into an eyepiece. LV diameter (LVD) and LV diameter adjusted (LVDA; LV wall width×LV diameter/mean sham LV diameter) were determined.
Coimmunoprecipitation and Western Blot Analysis
Homogenates of the LV myocardium were centrifuged, and the supernatants were resolved on 10% or 12% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. The membranes were incubated with primary monoclonal antibodies (Transduction Laboratories): eNOS, nNOS, Hsp90 (1:1000), caveolin-3 (1:500), and flotillin-1 (1:250). Protein loading was controlled using GAPDH antibody (1:5000; Ambion). In the coimmunoprecipitation experiments, the supernatant was incubated with monoclonal anti-eNOS after immunoprecipitation with protein A Sepharose (Zymed Laboratories). The immunoprecipitated protein was loaded and immunoblotted with monoclonal anti-Hsp90 to detect eNOS-bound Hsp90. The densitometric intensity was quantified by Quantity One software (BioRad).
Determination of cGMP Content
cGMP content in the LV tissues was measured using an acetylation protocol of a competitive enzyme-immunoassay system (Amersham Biosciences). The mean value was calculated from duplicate measurements of each sample and normalized per milligram of LV wet weight.
Purification of Caveolin-Enriched Membrane Fractions
Caveolae-enriched membrane fractions were prepared from isolated rat cardiomyocytes,13 according to a detergent-free purification method adapted from Song et al.15 Caveolin-3–enriched fractions obtained from each experimental group were loaded onto an electrophoresis gel and subjected to Western blot analysis as described above.
Data are presented as mean±SEM. Statistical significance was evaluated by analysis of Student’s t test or ANOVA followed by Newman-Keuls multiple comparisons test, when appropriate. Differences with values of P<0.05 were considered significant.
LA419 Chronic Treatment Attenuates Cardiac Hypertrophy Development and Abolishes the Presence of Fibrosis Without Changes in Blood Pressure
Heart weight (HW), HW:body weight ratio (HW:BW), LVD, LVDA, and mean arterial pressure were higher in the clip and clip+vehicle groups (7 and 14 weeks after aortic stenosis) than in the sham and sham+vehicle groups, respectively. Moreover, LVD and LVDA were significantly higher in the clip+vehicle group compared with the clip group, confirming the progression of LVH. Treatment for 7 weeks with the NO donor LA419, once cardiac hypertrophy was established, showed a marked reduction in the HW, HW:BW, LVD, and LVDA in the clip+LA419 group compared with the clip+vehicle group. There was no statistical difference in HW, LVD, and LVDA between the clip+LA419 and clip groups (euthanized 14 and 7 weeks after the surgery, respectively), which confirmed that LA419 treatment prevented the progression of LVH. Moreover, all of these effects of LA419 treatment were carried out without modifying the mean arterial pressure, which was significantly higher when comparing the clip+LA419 group with the sham+vehicle group (Table).
Histological examination in Figure 2 confirmed the extent of cardiac hypertrophy, with an increase of the LV cavity (Figure 2A) and cardiomyocyte size (Figure 2B and 2C) in the clip+vehicle group compared with the sham+vehicle group. Histological examination of the sham+LA419 group showed no difference compared with the sham+vehicle group. LA419 treatment reduced the size of LV (Figure 2A), as well as the size of cardiomyocytes (Figure 2B and 2C). Moreover, the area of enzymatically dispersed cardiomyocytes was measured, and it showed values of 8719±318 μm2 in the sham+vehicle group and 11 450±334 μm2 in the clip+vehicle group (P<0.001). After LA419 treatment, the myocyte area was reduced to 10 260±279 μm2 in the clip+LA419 compared with clip+vehicle groups (P<0.01). Interestingly, 3 of 7 hearts (43%) from the clip+vehicle group showed focal areas of interstitial (Figure 2D) and perivascular fibrosis (Figure 2E and 2F). However, these pathological collagen depositions were not observed in the clip group 7 weeks after the surgery. Histological and quantitative analysis confirmed that none of 13 hearts from the clip+LA419 group showed areas of pathological fibrosis (Figure 2D through 2F).
Effect of Pressure Overload and LA419 Treatment on Constitutive NO Synthase Expression and Hsp90-eNOS Association
Figure 3A shows that eNOS protein expression in LV samples obtained from animals 7 weeks after stenosis was reduced (40% of reduction in the clip versus sham group). This decrease in eNOS abundance was even more prominent after 14 weeks of pressure overload (69% of expression reduced in the clip+vehicle versus sham+vehicle groups; Figure 3B). LA419 treatment induced an important recovery of eNOS expression in the clip+LA419 group (Figure 3B). Analysis of nNOS protein expression (Figure 3C and 3D) did not show any differences among the groups.
Figure 4A and 4B show that protein expression of the chaperone Hsp90 was increased in heart extracts obtained from the clip and clip+vehicle groups (7 and 14 weeks after the surgery) compared with sham groups. LA419 did not modify the increased Hsp90 protein levels. To test the interaction between eNOS and its positive regulator Hsp90, we immunoprecipitated eNOS from LV homogenates and immunoblotted for Hsp90 protein. Although the total amount of eNOS protein in LV homogenates showed a decrease (Figure 3A and 3B), the immunoprecipitation procedure ensured equal loading of eNOS in samples from either experimental group. Figure 4C shows that the amount of Hsp90 bound to eNOS was decreased in the clip and clip+vehicle groups compared with the sham or sham+vehicle groups. LA419 treatment markedly improved the association of Hsp90 with eNOS in the clip+LA419 group.
cGMP Contents in Heart Extracts
Figure 5 shows that cardiac cGMP levels were decreased in the clip and clip+vehicle groups compared with the sham and sham+vehicle groups (Figure 5A and 5B). Chronic treatment with the NO donor LA419 showed no variation in cGMP cardiac levels in the sham+LA419 compared with sham+vehicle groups. Although NO released from LA419 was not able to increase cGMP cardiac content at basal condition, treatment with this NO donor was able to increase the cGMP cardiac levels in the clip+LA419 compared with clip+vehicle groups (Figure 5B).
Pressure Overload and LA419 Treatment Regulates Caveolin-3 Expression
We prepared a caveolae-enriched membrane fraction using a discontinuous sucrose density gradient. We detected the majority of caveolin-3 and flotillin-1 (another marker of caveolae) in fraction number 5 of the gradient (Figure 6A). Caveolin-3–enriched fraction 5 of cardiomyocyte fractionations obtained from each experimental group was loaded on a preparative gel, and identical protein concentration from fraction 5 was subjected to SDS-PAGE. The amount of caveolin-3 expression obtained in the clip group was 2 times higher than that obtained in the sham group (Figure 6B). However, the clip+vehicle group showed an important decrease of caveolin-3 levels compared with the sham+vehicle group (Figure 6C). Interestingly, we observed the same pattern of the expression for eNOS compared with caveolin-3 expression in the caveolin/flotillin fraction 5 at 14 weeks after the surgery (Figure 6D). Treatment of stenosed animals with LA419 restored the levels of expression to control values in both proteins, caveolin-3 and eNOS (Figure 6C and 6D).
We have demonstrated that chronic LA419 treatment prevented the progression to maladaptative cardiac remodeling through a mechanism that involved restoration of cardiac eNOS protein levels, the normalization of the molecular association with its allosteric regulator Hsp90, the re-establishment of caveolin-3 abundance, and the normal tissular content of cGMP. In the present study, we used an experimental progressive model of LVH induced by abdominal aortic stenosis. Stenosed rats showed a transition from a state of adaptative to maladaptative cardiac hypertrophy (from 7 to 14 weeks after stenosis), characterized by an increase in the LVD and LV cavity, and the presence of interstitial and perivascular fibrosis. The novel NO donor LA419 inhibited cardiac structural remodeling through the reversion of myocardial dilated hypertrophy and the prevention of the presence of pathological collagen deposition at the extracellular matrix level without modifying the blood pressure.
It is well known that cardiac hypertrophy development is controlled by counterregulatory signaling pathways, where NO exerts a potent antihypertrophic effect.16 In this sense, previous studies have reported a selective decrease of eNOS cardiac protein in different models of cardiac hypertrophy.17,18 The recent generation of transgenic mice models with a lack or an overexpression of eNOS protein has also demonstrated the importance of this enzyme in the process of cardiac remodeling.19,20 More recently, Wenzel et al21 have demonstrated that inhibition of eNOS-derived NO induces hypertrophy development in adult ventricular cardiomyocytes. All of this evidence indicates that NO produced by eNOS protects the heart from adverse remodeling. In the present study, we have demonstrated that the decrease in the protein level of eNOS induced by cardiac hypertrophy was re-established by the treatment with LA419. The amount of NO produced by eNOS is not only dependent on the enzyme abundance but is also regulated through posttranslational mechanisms, including the interaction with its allosteric regulators, such as Hsp90 and caveolin-318 (the enzyme’s stimulatory and inhibitory interactions, respectively). In our study, the decreased levels of eNOS contrasted with the augmented levels of Hsp90 in both states of LVH. These results could be the consequence of a compensatory mechanism to enhance the ability of eNOS to produce NO in stenosed rats, which showed low levels of eNOS expression. However, this compensatory mechanism might be useless, because the amount of Hsp90 bound to eNOS was decreased in the presence of LVH. LA419 treatment enhanced the interaction between Hsp90 and eNOS without modifying the increased Hsp90 protein levels in stenosed rats. Therefore, these results indicate that re-establishment of physiological eNOS abundance and increased association of eNOS with Hsp90 after LA419 treatment could enhance the endogenous ability of eNOS to produce NO in the stenosed animals. A potential explanation for these results could be related to the increase in the Hsp90 expression with hypertension22 or with increasing levels of angiotensin II.23 It is well known that angiotensin II has a key role in the process of myocardial remodeling, being directly involved in the induction of cardiac fibrosis.24 In our study, stenosed animals treated or not treated with LA419 showed a significant elevation of plasma angiotensin II values compared with sham nonstenosed animals (data not shown). However, histological analyses demonstrated the absence of pathological collagen deposition in stenosed animals treated with LA419. That fact could be explained by the antifibrotic effect of the NO at the cardiac extracellular matrix level, without excluding the possibility of a direct LA419 effect on local renin-angiotensin system in the heart.
It is well known that after NO synthesis and subsequent activation of the enzyme soluble guanylate cyclase, the immediate second messenger is cGMP. Different lines of evidence demonstrate that cGMP is an important cardioprotective agent against cardiac hypertrophy development.25 In the present study, stenosed rats showed a decrease in cGMP content that was re-established after LA419 treatment. This re-establishment in the cardiac cGMP levels could interfere with the signaling cascades that are functionally important in the context of cardiac hypertrophy and prevent the progress to maladaptive cardiac hypertrophy. Interestingly, LA419 was not able to modify the cardiac cGMP levels at basal condition. These results, together with the previous observation that LA419 infusion did not increase cGMP plasma levels,12 support the idea that bioactive NO released by LA419 was not high enough to activate soluble guanylate cyclase. Therefore, the normal tissular cGMP could be re-established as a consequence of restored normal eNOS expression in stenosed rats treated with LA419 and not as a consequence of the direct LA419 effect on soluble guanylate cyclase.
Caveolins, the structural proteins of the caveolae domain, modulate numerous signaling pathways, including NO production. Moreover, changes in caveolin-3 expression cause changes in the number of caveolae26 and consequently in NO synthase activity.27 In this sense, it has been proposed recently that when the abundance of caveolin is increased, eNOS is inactivated by excess inhibitory clamping. Interestingly, in cells lacking caveolae (or with a reduced amount of caveolin), the coupling between the agonist-bound receptor and cytosolic eNOS is lost, and NO production is also decreased.28 Our study demonstrates that the abundance of caveolin-3 varies with LVH development and is higher in clip rats after 7 weeks of stenosis and dramatically lower in clip+vehicle rats after 14 weeks of pressure overload. Thus, these findings are consistent with the upregulation of caveolin-3 observed in hypertrophied cardiomyocytes induced by phenylephrine and pressure overload.29 On the other hand, caveolin-3 expression is decreased in the heart of spontaneously hypertensive rats18 and in dogs with hypertrophic cardiomyopathy.30 Moreover, transgenic studies in mice with deletion of caveolin-3 have demonstrated that loss of this protein is sufficient to induce the molecular program leading to hypertrophic cardiomyopathy.31 In the present study, LA419 treatment restored the normal caveolin-3 abundance in rats with aortic stenosis. These results suggest that the complete loss of caveolin-3 might modify the architecture of the caveolae domain and contribute to the progression to maladaptative cardiac hypertrophy. Moreover, this fact could induce the loss of eNOS from this microdomain, which is observed in our stenosed animals. Thus, normal levels in caveolin-3 expression could be an important condition to restore the physiological stage of caveolae domain and to regulate the antihypertrophic signaling cascades, such as the NO pathway.
Our data provided evidence that the chronic administration of LA419 inhibited dilated cardiac hypertrophy and myocardial fibrosis without modifying high arterial blood pressure in stenosed overloaded rats. These effects of LA419 treatment were mediated, at least in part, by the re-establishment of the eNOS signaling pathway.
The present study demonstrated the beneficial effects of the NO donor LA419 in preventing the progression to maladaptative cardiac hypertrophy in a well-characterized model of LVH by pressure overload. Its beneficial effects are blood pressure independent and mediated through the restoration of the endogenous NO pathway in rats subjected to aortic stenosis. LA419 has been designed to treat clinical conditions where there exists reduced bioavailability of endogenous NO without tolerance development at long term.32 Moreover, previous studies have confirmed important anti-ischemic, antithrombotic, and antiatherosclerotic properties10–12 of this NO donor at doses that do not modify arterial blood pressure. Thus, these effects, together with the antihypertrophic properties demonstrated in the present study, suggest that LA419 could be a potentially useful drug not only in the prevention of thrombotic and ischemic complications of cardiovascular diseases but also in the prevention of maladaptative cardiac remodeling in the human heart. Further studies with a comparative standard treatment (ie, angiotensin-converting enzyme inhibitors) would be necessary to provide conclusive evidence of the beneficial effects of LA419 in the clinical setting.
We are grateful to Ferrán Giménez, Manuel Bas, Fernando Ortego, and María Luisa Hidalgo for their excellent technical assistance. We also thank Drs Andrea Barbuti, Olivia Hurtado, Dolores Gutierrez-Lopez, and Alicia Monjas for helping us with the experimental protocols and Dr Angélica Rueda for careful reading of this article.
Sources of Funding
This study was supported by the Ministerio de Educación y Ciencia of Spain (BFI2002-00536 and SAF-2005-01887) and Lacer S.A. G.R-H. and M.F.-V. are graduate research fellows of the Ministerio de Educación y Ciencia, and Consejo Superior de Investigaciones Científicas of Spain, respectively. C.D. is a member of the Red Temática de Enfermedades Cardiovasculares (RECAVA).
The first 2 authors contributed equally to this work.
- Received May 8, 2007.
- Revision received May 29, 2007.
- Accepted October 12, 2007.
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